The invention relates to a light detector for use in a scanning microscope, and in particular in a line scanning microscope. Further, the invention relates to a microscope comprising the light detector according to the invention.
Scanning microscopes are used in very different fields of engineering and sciences. A basic principle of such scanning microscopes is that one or more microscope beams are deflected in one or more dimensions by means of a beam deflector arrangement in order to screen (scan) a sample.
Various versions of scanning microscopes are known, which differ, for instance, in the type and generation of the microscope beam. For example, use can be made of electromagnetic beams in the optical, infrared or ultraviolet range of the spectrum, or in other ranges of the electromagnetic spectrum, for instance in the radiographic range. Also other types of beams are possible, such as particle beams in the form of electron beams or in the form of charged or neutral particles. It is also conceivable to use several microscope beams at the same time.
Further differences between the various types of scanning microscopes result from the interaction of the microscope beam or microscope beams with the sample to be examined. In the following description reference is made primarily to fluorescence microscopes, whose microscope beam excites a sample fluorescence which can be detected and used for image acquisition. However, numerous further measuring principles exist, such as measuring principles based on laser-spectroscopic methods, measuring principles based on particle emission, and other measuring principles. The following invention can be basically applied to all such methods.
A basic problem, which plays a major role in many of the microscopes described above as well as in other image acquisition methods, is the signal-to-noise-ratio and/or the signal strength of the signals to be detected. In particular, in fluorescence microscopy the saturation of the colorants used becomes noticeable in this respect, which saturation limits the maximum obtainable signal per pixel. Depending on the scanning speed and image format, often not enough fluorescence photons are available. The periods of time during which a single pixel of a detector is illuminated in a scanning cycle are typically no longer than some few nano seconds to some few 10 nano seconds. For example in a resonant bidirectional scanning apparatus with a scanning speed of 16,000 lines per second and 1,024 points per line, typically 25 nano seconds are available per pixel in the center of the image. For a signal-to-noise ratio of 20, it would be necessary to detect 400 photons per pixel (shot noise). This corresponds to 1.6·1010 detected photons per second or, with an assumed wavelength of 600 nm (3.3·10−19 J/photon), to a fluorescence performance of the actually detected photons of 5.3 nW. Assuming that only one quarter of the photons coming from the sample arrive at the detector and that this detector detects only about one quarter of the incoming photons, a fluorescence performance of at least about 84.8 nW must be emitted by the sample. With an assumed fluorescence lifetime of 5·10−9 s this corresponds to 1,280 colorant molecules which have to emit permanently (saturatedly) in the focus area.
This arithmetic example shows that the signal-to-noise ratio is a basic problem in scanning microscopes since for procedural reasons only an extremely limited period of time is available for gathering light quantums for single pixels. Increasing this time period however entails increasing the time for image formation which involves inconveniences for a user and/or which can lead to blurring of the image in case of quickly changing samples.
The CCD chips (charge-coupled device) normally used as image detectors in microscopes further cause additional difficulties in the form of increased noise. For example in CCDs, in addition to shot noise, readout noise is often dominating especially at small signal levels, which noise as a rule increases even further as the speed increases. Moreover, in line scanners as compared to point scanners optical resolution is reduced. The speed depends above all on the frame rate of the camera. For the particularly sensitive EMCCDs the maximum pixel rates are at present at about 35 MHz, which at 1 k·1 k corresponds to a frame rate of 30 frames per second.
Other types of image detectors than CCD image detectors are also known in the art. So-called avalanche semiconductor diodes (avalanche photodiodes), which are based on the effect of internal charge amplification, are particularly well suited for the high speeds necessary for scanning microscopy. Avalanche semiconductor diodes (also abbreviated as APDs in the following) are highly sensitive, fast photodiodes which can for instance be silicon-based. Usual APDs make e.g. use of InGaAs—InP-multy layer constructions. APDs may be operated in a linear range, in which the current signal is at least approximately linear to the number of irradiated photons, and, above a breakdown voltage, in a non-linear range, in which range the avalanche effect mentioned above occurs and which is also referred to as Geiger range.
The use of APD detectors, including in an array-arrangement, has been suggested repeatedly also in the field of microscopy. For instance DE 10 2005 059 948 A1 describes a detector which can be used above all for the spectral detection of light in a microscope. In this document it is suggested, among other things, to provide the detector with an APD-array.
Also DE 10 2004 003 993 A1 describes the use of an APD-array. In this document it is suggested to spectrally split up light of a spatially delimited source and to scan the spectrum with the help of an APD array.
APD arrays can be produced by means of semiconductor production methods known to a person skilled in the art. One example of a corresponding production method is described in U.S. Pat. No. 4,458,260. In this document an APD array is described which comprises numerous p-n junctions. Various layer assemblies for APDs and APD arrays are known in the art.
Despite the various previous suggestions to use APD arrays in the field of microscopy, however, the use of such arrays still poses a technical challenge. One particular difficulty consists in acquiring the great volume of data in a suitable manner and to process it at a corresponding speed. Also in many cases the dead time, which is a known problem in APDs, leads to difficulties, since it further reduces the length of image time available for 1 pixel and increases fluctuations in the counting rates.